A mutation (eth) conferring resistance to ethionine, an analog of the amino acid methionine, in Bacillus subtilis has been studied. The eth mutation is pleiotropic, causing ethionine-resistance, increased spontaneous sporulation at 33 C, conversion of relA mutants from high to low serine-sensitivity, protection of phage phi105 grown in an eth strain against hsrR restriction and relaxed RNA synthesis as well as absence of ppGpp synthesis upon amino acid starvation. B. subtilis strains carrying the eth mutation continually enter sporulation at a much higher rate in the presence of 2mM DL-ethionine than in its absence. The fact that sporulation is caused by ethionine in a relaxed background suggests that in these mutants the onset of sporulation bypasses the GTP drop believed to be the initial event when sporulation is initiated by nutrient deprivation. In contrast to Escherichia coli, B. subtilis produces S-adenosylethionine (SAE) upon ethionine addition. Inclusion of a mutation causing a deficiency in S-adenosylmethionine (SAM) synthetase activity (metE1) to the eth background abolishes the increase in sporulation upon ethionine addition and prevents the synthesis of SAE. This finding suggests that ethionine causes sporulation via SAE by interfering with the methylation or causing the ethylation of some cellular component. In the eth mutant, DNA sites specific to the hsrR restriction endonuclease, are more methylated than in the standard strain. The DNA modification activity influenced by the eth marker is likely the methylase normally induced during competence for DNA uptake and subsequent transformation. Changes in methylation of the genetic material occur during differentiation in mammalian cells. Our studies are the first to show a similar phenomenon in the differentiation of a microorganism which is accessible to the full range of tools of molecular genetics and biochemistry.